Method for preparing substituted 3,7-dihydroxy steroids

ABSTRACT

The invention relates to processes for preparing 17-alkynyl-7-hydroxy-steroids, such as 17-Ethynyl-10R,13S-dimethyl 2,3,4,7,8R,9S,10,11,12,13,14S,15,16,17-hexadecahydro-1H-cyclopenta[a]phenanthrene-3R,7R,17S-triol (also referred to as 17α-ethynyl-androst-5-ene-3β,7β,17β-triol), that are essentially free of process impurities having binding activity at nuclear estrogen receptors.

CROSS-REFERENCE TO RELATED APPLICATIONS

This non-provisional U.S. patent application is a continuation of U.S.non-provisional application Ser. No. 13/664,304, filed Oct. 30, 2012 andissued Oct. 20, 2015 as U.S. Pat. No. 9,163,059, which is a divisionalapplication of U.S. non-provisional application Ser. No. 12/479,626,filed Jun. 5, 2009 and issued on Nov. 13, 2012 as U.S. Pat. No.8,309,746, which claims priority to U.S. provisional application Ser.No. 61/059,658, filed Jun. 6, 2008, now expired, all of which areincorporated herein by reference in their entireties.

FIELD OF THE INVENTION

Invention embodiments relate to methods for preparing17-Ethynyl-10R,13S-dimethyl2,3,4,7,8R,9S,10,11,12,13,14S,15,16,17-hexadecahydro-1H-cyclopenta[a]phenanthrene-3R,7R,17S-trioland other pharmaceutically active compounds related thereto, that areessentially free of reaction steroid impurities having undesired bindingactivity at sex steroid receptors.

BACKGROUND OF THE INVENTION

17-Ethynyl-10R,13S-dimethyl2,3,4,7,8R,9S,10,11,12,13,14S,15,16,17-hexadecahydro-1H-cyclopenta[a]phenanthrene-3R,7R,17S-triol(also referred to herein as 17α-ethynyl-androst-5-ene-3β,7β,17β-triol orCompound 1) is effective in treating conditions that are attributable tochronic non-productive inflammation. In contrast to otheranti-inflammatory steroids, Compound 1 has been found to be essentiallyfree of binding activity at nuclear sex steroid receptors, activitywhich can contribute to unwanted side effects from such compounds.Steroid synthetic intermediates, by-products, side products or othersuch impurities that can affect or modulate sex steroid receptoractivity(ies) and may be present in preparations of Compound 1 areundesirable, since they contribute to side effects. Thus, methods forpreparing Compound 1 and analogs and derivatives thereof that avoidproduction of impurities, such as synthetic intermediates, steroidside-products or byproducts that affect or modulate nuclear sex steroidactivity(ies) are useful.

SUMMARY OF THE INVENTION

Reaction sequences to provide 17α-alkynyl-androst-5-ene-3β,7β,17β-triolhave unexpectedly been found to produce material containing undesiredsteroid impurity(ies) that impart sex steroid receptor activity(ies)that otherwise would not be present to the material. These impuritiesadversely affect the pharmaceutical acceptability of the17α-alkynyl-androst-5-ene-3β,7β,17β-triol. The presence of theseimpurities in preparations of 17α-alkynyl-androst-5-ene-3β,7β,17β-triolhas not been described and their presence adds additional cost to removeor reduce their presence to a level at which the sex steroidactivity(ies) would not be present. The reaction sequences disclosedherein avoid production of the undesired steroid impurity(ies) and thusavoid the need for purification of the17α-alkynyl-androst-5-ene-3β,7β,17β-triol so produced to effect removalor reduction in level of the impurity(ies).

One embodiment of the invention provides a reaction sequence forintroducing an alkynyl group at position 17 and an oxygen functionalityat position 7 to an androst-5-ene having an oxygen linked group atposition 3 and a ketone at position 17 such that a side-product lackinga oxygen substituent at C-7, and thus having undesired binding activityat sex steroid receptors, is precluded.

Another embodiment of the invention provides a reaction sequence forintroducing an ethynyl group at position 17 and a hydroxy group atposition 7 to dehydroepiandrosterone (also referred herein as DHEA or3β-hydroxy-androst-5-ene-17-one) such that a preparation of Compound 1is formed that is essentially free of binding activity at sex steroidreceptors.

In another embodiment of the invention provides for a reaction sequencethat uses DHEA as starting material to obtain a preparation containingCompound 1 that is essentially free of the estrogenic compound17α-ethynyl-androst-5-ene-3β,17β-diol or its potential precursor17α-ethynyl-3β-acetoxy-androst-5-ene-17β-ol.

In one embodiment of the invention the reaction sequence comprises aprior step of oxidizing an appropriately protected androst-5-ene havinga first and second oxygen linked group at positions 3 and 17 such that athird oxygen linked group is introduced at position 7 and a subsequentstep of reacting an intermediate wherein the second oxygen linked groupat position 17 is ═O with an anion derived from an alkyne.

In another embodiment of the invention the reaction sequence comprises aprior step of oxidizing DHEA, appropriately protected, to introduce athird oxygen linked group at position 7 and a subsequent step ofreacting of an intermediate having the substituent ═O at position 17with an acetylene anion, optionally protected.

In another embodiment of the invention the reaction sequence comprisesthe step of reacting androst-5-en-17-one-3β,7β-diol, appropriatelyprotected, with an anion derived from an alkyne.

DETAILED DESCRIPTION Definitions

As used herein and unless otherwise stated or implied by context, termsthat are defined herein have the meanings that are specified. Thedescriptions of embodiments and examples that are described illustratethe invention and they are not intended to limit it in any way. Unlessotherwise contraindicated or implied, e.g., by including mutuallyexclusive elements or options, in these descriptions and throughout thisspecification, the terms “a” and “an” mean one or more and the term “or”means and/or.

“Alkyl” as used here refers to linked normal, secondary, tertiary orcyclic carbon atoms, i.e., linear, branched, cyclic or any combinationthereof. Alkyl groups or moieties, as used herein, may be saturated, orunsaturated, i.e., the moiety may comprise one, two, three or moreindependently selected double bonds or triple bonds. Unsaturated alkylmoieties include moieties as described below for alkenyl, alkynyl,cycloalkyl, and aryl moieties. The number of carbon atoms in an alkylmoiety is 1-20, preferably 1 to 8. C₁₋₈ alkyl or C1-8 alkyl means analkyl moiety containing 1, 2, 3, 4, 5, 6, 7 or 8 carbon atoms and C₁₋₆alkyl or C1-6 alkyl means an alkyl moiety containing 1, 2, 3, 4, 5 or 6carbon atoms. When an alkyl moiety is specified, species may includemethyl, ethyl, 1-propyl (n-propyl), 2-propyl (iso-propyl, —CH(CH₃)₂),1-butyl (n-butyl), 2-methyl-1-propyl (iso-butyl, —CH₂CH(CH₃)₂), 2-butyl(sec-butyl, —CH(CH₃)CH₂CH₃), 2-methyl-2-propyl (t-butyl, —C(CH₃)₃),1-pentyl (n-pentyl), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂) and 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃).

“Cycloalkyl” as used here refers to a monocyclic, bicyclic or tricyclicring system composed of only carbon atoms. The number of carbon atoms inan cycloalkyl group or moiety can vary and typically is 3 to about 20,e.g., preferably 3-8. C₃₋₈ alkyl or C3-C8 alkyl means an cycloalkylmoiety containing 3, 4, 5, 6, 7 or 8 carbon atoms and C₃₋₆ alkyl orC3-C6 means an cycloalkyl moiety containing 3, 4, 5 or 6 carbon atoms.Preferred cycloalkyl substituents are cyclopropyl, cyclopentyl,cyclohexyl, cycloheptyl and adamantyl. Cycloalkyl substituents having adouble bond within the cyclic ring system are sometimes referred to ascycloalkenyl substituents.

“Alkenyl” as used here means a moiety or group that comprises one ormore double bonds (—CH═CH—), e.g., 1, 2, 3, 4, 5, 6 or more, typically1, 2 or 3 and can include an aryl moiety such as benzene, andadditionally comprises linked normal, secondary, tertiary or cycliccarbon atoms, i.e., linear, branched, cyclic or any combination thereofunless the alkenyl moiety is vinyl (—CH═CH₂). An alkenyl moiety withmultiple double bonds may have the double bonds arranged contiguously(i.e., a 1,3 butadienyl moiety) or non-contiguously with one or moreintervening saturated carbon atoms or a combination thereof, providedthat a cyclic, contiguous arrangement of double bonds do not form acyclically conjugated system of 4n+2 electrons (i.e., aromatic). Thenumber of carbon atoms in an alkenyl moiety can is 2-20, preferably 2-8.C₂₋₈ alkenyl or C2-8 alkenyl means an alkenyl moiety containing 2, 3, 4,5, 6, 7 or 8 carbon atoms and C₂₋₆ alkenyl or C₂₋₆ alkenyl means analkenyl moiety containing 2, 3, 4, 5 or 6 carbon atoms. When an alkenylmoiety is specified, species include, e.g., any of the alkyl moietiesdescribed above that has one or more double bonds such as methylene(═CH₂), methylmethylene (═CH—CH₃), ethylmethylene (═CH—CH₂—CH₃),propylmethylenes (═CH—CH₂—CH₂—CH₃), vinyl (—CH═CH₂), allyl (—CH═CHCH₃),1-methylvinyl, butenyl, iso-butenyl, 3-methyl-2-butenyl or 1-pentenyl.

“Alkynyl” as used herein refers to linked normal, secondary, tertiary orcyclic carbon atoms where one or more triple bonds (—C≡O—) are present,typically 1, 2 or 3, usually 1, optionally comprising 1, 2, 3, 4, 5, 6or more double bonds, with the remaining bonds (if present) being singlebonds and comprising linked normal, secondary, tertiary or cyclic carbonatoms, i.e., linear, branched, cyclic or any combination thereof, unlessthe alkynyl moiety is ethynyl. The number of carbon atoms in an alkynylgroup or moiety is 2 to 20, preferably 2-8. C₂₋₈ alkynyl or C2-8 alkynylmeans an alkynyl moiety containing 2, 3, 4, 5, 6, 7 or 8 carbon atoms.When an alkynyl substituent is specified, preferred species include,—C≡CH, —C≡CCH₃, —C≡CCH₂CH₃, —C≡CC₃H₇ and —C≡CCH₂C₃H₇. Particularlypreferred species are ethynyl, propynyl and 1-butynyl with ethynylespecially preferred.

“Aryl” as used herein refers to an aromatic ring system or a fused ringsystem with no ring heteroatoms comprising 1, 2, 3 or 4 to 6 rings,typically 1 to 3 rings; wherein the rings are composed of only carbonatoms; and refers to a cyclically conjugated system of 4n+2 electrons(Hückel rule), typically 6, 10 or 14 electrons some of which mayadditionally participate in exocyclic conjugation (cross-conjugated).When an aryl group is specified, species may include phenyl, biphenyl,naphthyl, phenanthryl and quinone.

“Heteroaryl” as used here refers means an aryl ring system wherein oneor more, typically 1, 2 or 3, but not all of the carbon atoms comprisingthe aryl ring system are replaced by a heteroatom which is an atom otherthan carbon, including, N, O, S, Se, B, Si, P, typically, oxygen (—O—),nitrogen (—NX—) or sulfur (—S—) where X is —H, a protecting group orC₁₋₆ optionally substituted alkyl, wherein the heteroatom participatesin the conjugated system either through pi-bonding with an adjacent atomin the ring system or through a lone pair of electrons on the heteroatomand may be optionally substituted on one or more carbons or heteroatoms,or a combination of both, comprising the heterocycle in a manner whichretains the cyclically conjugated system.

“Protecting group” as used here means a moiety that prevents or reducesthe atom or functional group to which it is linked from participating inunwanted reactions. For example, for —O≡R^(PR), R^(PR) is a protectinggroup for the oxygen atom found in a hydroxyl, while for ═O theprotecting group is a ketal or thioketal wherein the divalent oxygen isreplaced, for example, by —X—(CH₂)_(n)—Y—, wherein X and Y independentlyare S and O and n is 2 to 3, to form a spiro ring system or an oximewherein the divalent oxygen is replaced by ═N—OR, wherein R is —H, alkylor aryl. For —C(O)—OR^(PR), R^(PR) is a carbonyloxy protecting group,for —SR^(PR), R^(PR) is a protecting group for sulfur in thiols forinstance, and for —NHR^(PR) or —N(R^(PR))₂—, R^(PR) is a nitrogen atomprotecting group for primary or secondary amines. The protecting groupsfor sulfur or nitrogen or monovalent oxygen atoms are usually used toprevent unwanted reactions with electrophilic compounds. The protectinggroups for divalent oxygen atoms (i.e. ═O) are usually used to preventunwanted reactions with nucleophilic compounds.

“Optionally substituted alkyl”, “optionally substituted alkenyl”,“optionally substituted alkynyl”, “optionally substituted heterocycle”,“optionally substituted aryl”, “optionally substituted heteroaryl” andthe like mean an alkyl, alkenyl, alkynyl, aryl, heteroaryl or othergroup or moiety as defined or disclosed herein that has a substituent(s)that optionally replaces a hydrogen atom(s). Such substituents are asdescribed above. For a phenyl moiety (-Ph), the arrangement of any twosubstituents present on the aromatic ring can be ortho (o), meta (m), orpara (p) to each other. Preferred optionally substituted moieties are—CF₃, —CH₂OH, —C≡C—Cl and -Ph-F.

“O-linked group”, O-linked substituent and like terms as used hereinrefers to a group or substituent that is attached to a moiety directlythough an oxygen atom of the group or substituent. An O-linked group maybe monovalent including groups such as —OH, acetoxy, i.e., —O—C(O)—CH₃,acyloxy, i.e., —O—C(O)—R wherein R is —H, optionally substituted alkyl,optionally substituted cycloalkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl, optionallysubstituted heteroaryl or optionally substituted heterocycle, aryloxy(Aryl-O—), phenoxy (Ph-O—), heteroaryloxy (Heteroaryl-O—), silyloxy,i.e. R₃SiO— wherein R independently are alkyl or aryl, optionallysubstituted or —OR^(PR) wherein R^(PR) is a protecting group aspreviously defined or may be divalent, i.e. ═O.

The term “preparation of Compound 1” refers to material preparedaccording to any one of the synthetic schemes specifically orgenerically described and includes Compound 1 wherein Compound 1 isfound as the major component on a mass basis and process impurities,present either in Compound when initially isolated as a solid or afterrecrystallization and-or purification of the solid.

The term “binding activity” refers to the ability of a specifiedcompound, a preparation comprising Compound 1 or impurity (orimpurities) in a preparation of Compound 1 to bind to or associate witha receptor, typically a sex steroid receptor such as an androgenreceptor or an estrogen receptor to effect or modulate the receptor'sbiological activity. Binding is usually measured in assays as thecapacity of a compound, usually an impurity in a preparation comprisingCompound 1, to displace a physiologically relevant ligand of a receptor(i.e., reference ligand) that is bound to that receptor in a competitionassay. The reference ligand is typically a natural ligand of thereceptor or an agonist of the receptor that has been labeled with aradioactive or spectroscopic probe whose presence may be queried byscintillation counting or by a spectroscopic method such as fluorescenceemission or fluorescence polarization.

The radioactive probe is typically ³H and/or ¹⁴C, where radioactiveatom(s) have replaced one or more of atoms of the ligand at positionswhere loss of the radiolabel would not occur to an extent underconditions of the assay that would complicate or confound interpretationof the assay results. The spectroscopic probe is typically a fluorophorethat is attached to a reference ligand at a position that provides alabeled reference ligand that has a K_(d) in the range of 0.1-100 nM atpositions and will not lose the fluorescent label to an extent underconditions of the assay that would complicate or confound interpretationof the assay results. The binding activity of the specified compound ora preparation of Compound 1 is typically expressed by K_(i), whereinK_(i) is determined from the concentration the specified compound orpreparation to displace the labeled reference ligand to the receptor by50% and the Kd of the labeled reference ligand.

“Sex steroid receptor” refers to nuclear receptors normally associatedwith affecting the growth or function of the reproductive organs and thedevelopment of secondary sex characteristics and includes androgenreceptor, estrogen receptor-α (ERα), estrogen receptor-β (ERβ) andprogesterone receptor.

“Essentially free” as used herein refers to a property of or an impurityin a preparation of Compound 1 as not being present or measurable in anamount that would adversely affect or detract from the desiredpharmacological activity of Compound 1. For example, the term“essentially free of sex steroid receptor binding activity” refers tothe absence of receptor binding activity for a preparation of Compound 1to nuclear sex steroid receptors as defined by values of K_(i)>10 μM forbinding to those receptors, as determined using standard receptorbinding assay conditions, and is irrespective of the identity of theimpurity that may be present in the preparation that would give rise tothe sex receptor binding activity. Likewise, essentially free ofestrogen receptor binding activity estrogen receptors refers to theabsence of receptor binding activity for a preparation of Compound 1 tonuclear estrogen receptors ERα and ERβ as defined by values of K_(i)>10μM for binding to those receptors, as determined using standard receptorbinding assay conditions, and is irrespective of the identity of theimpurity that may be present in the preparation that would give rise tothe estrogen receptor binding activity. When the term “essentially free”is used to describe the amount of an impurity present in a preparationof Compound 1, the term means the impurity is not present in an amountthat would adversely affect the pharmacological activity of Compound 1for its intended use by contributing to side effects normallyattributable to activation of nuclear estrogen receptor that are due tobinding activity of the impurity at these receptors. The impurity (e.g.,17α-ethynyl-androst-5-ene-3β,17β-diol) may directly affect thepharmacological activity of Compound 1 by binding or modulating theestrogen receptor or may indirectly affect the pharmacological activityof Compound 1 by its conversion in a subject being treated with acomposition comprising a preparation of Compound 1 by hydrolysis (eitherspontaneously or enzymatically) to a compound that does affect ormodulate the estrogen receptor (e.g.,17α-ethynyl-3β-acetoxy-androst-5-ene-17β-ol converting to17α-ethynyl-androst-5-ene-3β,17β-ol).

The terms “impurity” or “process impurity” as used herein refers to acomponent in a preparation of Compound 1 that is a steroid byproduct,side-product or a degradation product formed during synthesis ofCompound 1 and represents a minority contribution to the overall mass ofthe preparation, typically less than about 2%.

“Formulation” or “pharmaceutically acceptable formulation” as usedherein refers to a composition comprising a preparation of Compound 1,and one or more pharmaceutically acceptable excipients.

The inventions described herein provide for methods of preparing17-alkynyl steroids having oxygen substituents at positions 3, 7 and 17that are essentially free of impurities that have undesired bindingactivity at sex steroid receptors. Such 17-alkynyl steroids so preparedare essentially free of one or more impurities that are characterized bylacking the oxygen substituent at C7, which are responsible for theundesired binding activity.

The present methods were developed in response to unexpected estrogeniceffects seen with Compound 1. Compound 1 was prepared by the routecomprising the following steps referred to as Process A:

(1) Contacting a suitably protected acetylene anion with suitablyprotected dehydroepiandrosterone to introduce the ethynyl group atposition 17α by addition of the acetylene anion to the ═O functionalgroup at position 17;

(2) Contacting suitably protected 17α-ethynyl-androst-5-ene-3β,17α-diolwith an oxidizing agent to introduce the ═O functional group at position7; and

(3) Contacting suitably protected17α-ethynyl-androst-5-en-7-one-3β,17α-diol with a reducing agent todirectly convert the ═O functional group at position 7 to β-hydroxyl.

An embodiment of Process A is given in Scheme I (herein referred to asProcess A, Route 1). Compound 1 so produced was unexpectedly found tohave estrogenic effects. Impurity profiling of Compound 1 prepared bythis route showed the presence of 17α-ethynyl-androst-5-ene-3β,17β-diol,which has the same structure as Compound 1 except the β-hydroxy group atposition 7 is absent. Receptor binding studies showed that17α-ethynyl-androst-5-ene-3β,17β-diol had significant binding activityat the estrogen receptors ERα and ERβ, whereas Compound 1 essentiallyfree of this impurity possessed no activity at these receptors whentested to 10 μM. Based upon this undesired sex steroid activity, a newprocess, referred to as Process B, was developed that circumventedproduction of the estrogenic side product 17α-ethynyl-androst-5-ene-3β,17β-diol.

Process B comprises the following steps.

(a) Contacting a suitably protected dehydroepiandrosterone with anoxidizing agent to directly introduce the ═O functional group atposition 7;

(b) Contacting a suitably protected androst-5-en-7,17-dione-3β-ol with areducing agent to convert the ═O functional group at position 7 toβ-hydroxyl;

(c) Contacting a suitably protected acetylene anion with suitablyprotected androst-5-en-17-one-3β,7β-diol to introduce the ethynyl groupat position 17α by addition of the acetylene anion to the ═O functionalgroup at position 17.

Procedures to effect step (a) include microbial oxidation as describedin Wuts, P. G. M. “A chemobiological synthesis of eplerenone” Synlett(3): 418-422 (2008); oxidation with oxo-chromium based reagents [e.g.,see Koutsourea, et al., “Synthetic approaches to the synthesis of acytostatic steroidal B-D bilactam” Steroids 68: 569-666 (2003) andCondom, et al., “Preparation of steroid-antigens through positions ofthe steroid not bearing functional groups” Steroids 23: 483-498 (1974)],peroxide assisted allylic oxidation [e.g., see Marwah, P., et al. “Aneconomical and green approach for the oxidation of olefins to eneones”Green Chem. 6: 570-577 (2004) and Marwah, P., et al., “Ergosteroids IV:synthesis and biological activity of steroid glucuronosides, ethers andalkylcarbonates” Steroids 66: 581-595 (2001)], oxidation withN-hydroxysuccimimide/AIBN [e.g., see Lardy, et al. “Ergosteroids II:Biologically active metabolites and synthesis derivatives ofdehydroepiandrosterone” Steroids 63:158-165 (1998)].

For step (a), a suitably protected dehydroepiandrosterone has the3β-hydroxyl and ═O functional groups protected with protecting groupstypically employed for hydroxyl and ketone given in Greene, T. W.“Protecting groups in organic synthesis” Academic Press, 1981. Thehydroxy protecting group should be suitable for conditions required to(1) protect the ═O functional group at position 17, if not alreadypresent, or (1′) does not require conditions to introduce that adverselyeffect a ketone protecting group previously introduced to position 17,and (2) introduce directly the ═O functional group at position 7.Preferred hydroxy protecting groups are also suitable for conditionsrequired to (3) reduce the ═O functional group to be introduced atposition 7 with sufficient selectivity to provide 78β-hydroxy as thepredominant isomer and (4) removed under conditions where an allylalcohol that is formed by the ═O functional group reduction hassufficient stability. Hydroxy protecting groups meetings conditions(1)-(4) include ester, usually aryl ester or C1-6 alkyl ester, when theprotecting group for the ═O functional group at position 17 is ketal andthe reducing agent to be used is a borohydride based reducing agent. Useof a stronger hydride reducing agent would require a hindered ester orsubstituted methyl ether as the hydroxy protecting group to preventpremature loss of the hydroxy protecting group. Preferred ═O protectinggroups are ketal, such as dimethyl ketal, diethyl ketal or the spiroketal prepared from ethylene glycol. A preferred suitably protecteddehydroepiandrosterone is the ethylene ketal of3β-acetoxy-androst-5-en-17-one.

Procedures to effect step (b) include reduction with metal hydride basedreagents such as the borohydride based reagents that include Zn(BH₄)₂,NaBH₄, optionally with a transition metal salt such as CeCl₃, NiCl₂,CoCl₂ or CuCl₂, L-Selectride (lithium tri-sec-butylborohydride) orN-Selectride (sodium tri-sec-butylborohydride). Lithium aluminum hydridebased or sodium aluminum hydride reagents may also be used althoughselectivity may suffer due to the reducing strength of such reagents.This may be ameliorated by using lithium aluminum hydride based reagentshaving alkoxy ligands to aluminum to reduce reactivity. Such reagentshave the general formula LiAl—H_(n) (OR)_(4-n), where n=1, 2, 3, R isC1-6 alkyl and include LTMA (lithium triethoxyaluminum hydride LTEAH(lithium triethoxyaluminum hydride), RED-AL (Sodiumbis(2-methoxyethoxy)aluminium hydride). Reduction using borohydridebased reagents may be conducted in alcohol solvents whereas reductionswith aluminium hydride based reagents require an ether solvent such asTHF. Selectivity may be improved, particularly for the aluminum hydridereagents, by conducting the reaction at temperature of between 0° C. to−78° C. with lower temperatures being more suitable for the aluminumhydride reagents.

Procedures to effect step (c) include in situ preparation of anacetylene anion followed by contact of the acetylide so formed with asuitably protected androst-5-en-17-one-3β,7β,17β-triol. The acetylidemay be prepared by contacting acetylene with an amide anion (e.g.,NaNH₂) in a hydrocarbon solvent such as benzene, toluene or xylene, asfor example in U.S. Pat. No. 2,251,939, with sodium or potassium metalin liquid ammonia, as for example in U.S. Pat. No. 2,267,257, or bycontacting a mono-silyl protected acetylene such as trimethylsilylacetylene with an organolithium reagent. Suitable organolithium reagentsinclude commercially available n-butyl lithium, sec-butyl lithium,methyl lithium, t-butyl lithium or phenyl lithium or can be prepared byreaction of an alkyl or aryl bromide with metallic lithium in an inertsolvent such as diethyl ether or tetrahydrofuran. The acetylide soprepared is then contacted with a suitably protectedandrost-5-en-17-one-3β,17β-diol.

For step (c), a suitably protected androst-5-en-17-one-3β,7β-diol willhave hydroxyl protecting groups that are typically used in carbanionchemistry and may be removed under conditions that are compatible withthe presence of a terminal alkyne and an allylic alcohol and includeprotecting groups that are removed under neutral or mildly acidicconditions, typically between pH 3-7 and can be introduced underconditions compatible with an allylic alcohol. Preferred protectinggroups are silyl ethers of the formula (R¹)₃SiO— wherein R¹independently are aryl or C1-6 alkyl and include trimethylsilyl,triethylsilyl, t-butyldimethylsilyl, isopropyldimethylsilyl,t-butyldiphenylsilyl, methyldiisopropylsilyl, methyl-t-butylsilyl,tribenzylsilyl and triphenylsilyl ether. Preferred silyl ethers aretrimethylsilyl ether and t-butyldimethylsilyl ether. Some substitutedmethyl ethers may be used and include 2-(trimethylsilyl)-ethoxymethylether (SEM ether), tetrahydropyranyl ether (THP ether),tetrahydrothiopyranyl ether, 4-methoxy-tetrahydropyranyl ether,4-methoxytetrahydrothiopyranyl ether, tetrahydrofuranyl ether andtetrahydrothiofuranyl ether. Some substituted ethyl ethers that may beused as hydroxy protecting groups and include 1-ethoxyethyl ether andt-butyl ether. Preferred hydroxy protecting groups have lower stericdemands, such as trimethylsilyl ether and allow for simultaneousprotection of the 3β- and 7β-hydroxy groups.

Other procedures to effect step (c) include contacting a suitablyprotected androst-5-en-17-one-3β,7β-diol with sodium acetylide, lithiumacetylide (as its ethylene diamine complex), ethynyl magnesium halide(e.g., chloride or bromide) or ethynyl zinc halide, as for example inU.S. Pat. No. 2,243,887, in diethylether or other ether solvents such astetrahydrofuran, 1,2-dimethoxyethane, 2-methoxyethylether and the like.

One embodiment of Process B uses3β-acetoxy-androst-5-en-7-one-17,17-ethylenedioxy as the suitablyprotected androst-5-en-7,17-dione-3β-ol (see Scheme II; herein referredto as Process B, Route 1).

Another embodiment of Process B uses3β-acetoxy-androst-5-en-7-on-17-oxime as the suitably protectedandrost-5-en-7,17-dione-3β-ol (see Scheme III; herein referred to asProcess B, Route 2)).

The following examples and schemes further illustrate the invention andthey are not intended to limit it in any way.

EXAMPLES Example 1

Synthesis of 3β-trimethylsilyloxy-androst-5-en-17-one (TMS-DHEA): DHEAis combined with 1,1,1,3,3,3-hexamethyldisilazane (HMDS) and saccharin(as catalyst) in acetonitrile. The reaction mixture was heated to refluxfor several hours with stirring under a nitrogen atmosphere. Liberatedammonia was purged under slight vacuum. The volume was then reduced bydistillation, followed by cooling the mixture and collecting theprecipitated product by filtration. The filter cake of TMS-DHEA productwas washed with cold acetonitrile and dried with warm nitrogen to a losson drying (LOD) of not more than 0.5% to provide approximately 90%isolated yield of the title compound.

Example 2

Synthesis of 17α-ethynyl-androst-5-ene-3β,17β-diol: n-Butyl lithium isadded slowly to Me₃Si—C≡CH in THF under a nitrogen atmosphere atapproximately 0° C. to produce the lithium acetylide Me₃Si—C≡C—Li. Thetemperature was raised to about 20° C., and TMS-DHEA was added as asolution in THF, and stirred for about 3 hours. The reaction wasquenched by raising the temperature to about 40° C., followed by theslow addition of methanol. Liberated acetylene is purged under slightvacuum. Concentrated KOH was then slowly added until gas evolutionsubsides, and the volume was reduced by approximately 50% by vacuumdistillation at approximately 45° C. Excess 6 N HCl was slowly added,while maintaining the temperature at approximately 40° C. The reactionmixture was diluted with water and chilled to approximately 5° C. beforecollecting the product by filtration and washing the filter cake withcold 50/50 methanol water. The product was dried with warm nitrogen toan LOD of not more than to 0.5% to provide approximately 87% isolatedyield of the title compound.

Example 3

Synthesis of 17α-ethynyl-3β-acetoxy-androst-5-en-17β-ol:17α-ethynyl-androst-5-ene-3β,17β-diol was mixed with acetic anhydride,triethylamine, and a catalytic amount of DMAP in THF at refluxingtemperature for at least 4 hours. The reaction progress was monitored byHPLC, and allowed to proceed until not more than 1% of the startingmaterial remains. The mixture was then cooled to 30-50° C., and waterwas added, followed by cooling to approximately 0° C. for 1 hour. Thecrude product was collected by filtration, washed with coldacetonitrile, and dried with warm nitrogen to an LOD of not more than5%. Crude product was characterized by HPLC and recrystallized fromacetonitrile if the title compound was present in less than 95% peakarea purity by HPLC. The recrystallized product was collected byfiltration and dried to provide an overall isolated yield of 83% of thetitle compound.

Example 4

Synthesis of 17α-ethynyl-38-acetoxy-17β-ol-androst-5-en-7-one:17α-ethynyl-3β-acetoxy-androst-5-en-17β-ol was combined with t-butylhydroperoxide and copper (I) iodide in acetonitrile and refluxed for twohours. The reaction was then quenched by cooling to about 50° C.followed by adding a large excess of aqueous sodium thiosulfate solutionwith stirring for at least 30 minutes. Under these conditions, organicand aqueous phases are not miscible. Agitation was halted to allow phaseseparation, and the aqueous phase (lower) is drained. The organic phasewas extracted twice with aqueous sodium sulfite solution and brine bymixing at about 45° C. for at least 30 minutes, followed by phaseseparation, and removal of the aqueous phase. The resulting organicphase was concentrated to approximately 25% of the original volume byvacuum distillation at less than 45° C., and then chilled to about 5° C.The crude product, 17α-ethynyl-3β-acetoxyl-androst-5-en-7-one-17β-ol,was collected by filtration. The filtered cake was washed with coldacetonitrile/water and dried with warm nitrogen to an LOD of not morethan 1.0%. The crude product was recrystallized twice by dissolving inN-methyl-pyrrolidinone (NMP) at approximately 90° C., followed bycooling to 0° C. for approximately one hour. The title compound wascollected by filtration for an isolated overall yield of approximately30%.

Example 5

Synthesis of 17α-ethynyl-androst-5-ene-3β,7β,17β-triol:17α-ethynyl-3β-acetoxy-androst-5-en-7-one-17β-ol was reduced with NaBH₄in THF/methanol in the presence of CeCl₃ at approximately 0° C. forabout 2 hours. The reaction progress was monitored by HPLC. The reactionwas quenched by slow addition of dilute HCl with liberated hydrogenremoved under slight vacuum. Methyl tert-butyl ether (MTBE) was addedand the reaction mixture washed twice with brine, discarding the aqueousphases. The 3β-acetoxy group from the crude product was removed byaddition of methanolic KOH to the organic phase at approximately 0° C.for about 2 hours, with reaction progress monitored by HPLC. Aftercompletion, the reaction mixture was neutralized with acetic acid, andapproximately one-half of the reaction volume was removed by vacuumdistillation at less than 45° C. Approximately two-thirds of theoriginal reaction mixture volume was added as isopropanol (IPA), and thevolume was subsequently reduced to approximately one-fourth the originalvolume by vacuum distillation at less than 45° C. The residue mixturewas cooled to 0° C., and the crude17α-ethynyl-androst-5-ene-3β,7β,17β-triol was collected by filtration,washed with cold IPA, and dried with warm nitrogen.

Example 6

Recrystallization of 17α-ethynyl-androst-5-ene-3β,7β,17β-triol fromProcess A: Crude 17α-ethynyl-androst-5-ene-3β,7β,17β-triol from Example5 was dissolved in refluxing methanol/water (˜10/1). Methanol wasremoved by vacuum distillation while stirring, and replaced withsufficient water to maintain suspension of the crystallized product. Thesuspension is cooled to approximately 5° C. with stirring, and theproduct was recovered by filtration. The filtered cake was washed withwater and dried under vacuum to less than 0.5% water to provide thetitle compound in approximately 69% isolated yield.

Example 7

Synthesis of 3β-acetoxy-androst-5-ene-17,17-ethylenedioxy: A 300 Lreactor was charged with 36 kg of triethylorthoformate, 20 kg of3β-acetoxy-5-androsten-17-one, 12.6 kg of ethylene glycol and 400 g ofp-toluenesulfonic acid. The mixture was heated to reflux under nitrogenuntil the reaction was complete (about 2-3 hours). The mixture was thencooled to 60° C. and 16 kg of anhydrous ethanol and 400 ml of pyridinewere added. The resulting solution was transferred to a container andrefrigerated overnight. The solids that formed were filtered and washedwith 80 kg of 50% ethanol and dried at 40-50° C. to afford 18.5-21.0 kg(81.5-92.5%) of the title compound.

Example 8

Synthesis of 3β-acetoxy-androst-5-en-7-one-17,17-ethylenedioxy: A 500 Lreactor was charged with 200 kg ethyl acetate and 25 kg of3β-acetoxy-androst-5-en-17,17-ethylenedioxy. The mixture was stirred for30 minutes whereupon 55 kg of 70% t-butyl peroxide and 9 kg of sodiumbicarbonate were added. The reaction mixture was then cooled to 0° C.and 116 kg of 13% sodium perchlorate (aq.) was added over 10 hours sothat a reaction temperature below 5° C. and pH between 7.5 and 8.5 weremaintained. After the reaction was complete, the organic layer wasseparated and the aqueous phase was extracted with ethyl acetate (35kg×2). The combined organic phase was combined with a solution 33 kg ofsodium sulfite in 167 kg of water, and the resulting mixture was stirredat 40° C. for 3 hours. The organic phase was washed with 50 kg of brineand concentrated to 55-60 kg whereupon 50 kg of methanol was added.After refrigeration overnight, a white solid was formed that wasfiltered and washed with 10 kg of methanol, and dried at 40-50° C. toyield 7.1-7.8 kg (27.4-30.1%) of the title compound.

Example 9

Synthesis of 3β-acetoxy-androst-5-ene-17,17-ethylenedioxy-7β-ol. A 500 Lreactor was charged with 48 kg of THF, 10 kg of3β-acetoxy-androst-5-en-7-one-17,17-ethylenedioxy and a solution of 9.6kg CeCl₃.7H₂O in 95 kg methanol. This mixture was cooled to 0° C.whereupon 2.0 kg of NaBH₄ was added in batches over 3 hours in order tomaintain the temperature below 5° C. After stirring for 30 more minutes,28 kg of acetone was added slowly in order to maintain the temperaturebelow 5° C., with stirring continued for another 30 minutes. To themixture was added 240 kg water with stirring continued for 1 hour. Theorganic solvents were removed under vacuum and the residue was extractedwith ethyl acetate (100 kg+50 kg). The combined organic phase was washedwith brine. Solvent was then removed to provide 8.6-8.9 kg (85.1-88.1%)of the title compound.

Example 10

Synthesis of 3β-acetoxy-androst-5-en-17-one-7β-ol: A 500 L reactor wascharged with 315 kg of acetone and 18 kg of3β-acetoxy-androst-5-en-17,17-ethylenedioxy-7β-ol. The mixture wascooled to 5° C. and 2.34 kg of p-toluenesulfonic acid was added slowlyto maintain the temperature below 10° C. After stirring the mixture at8-15° C. for 36-48 hours, 3.0 kg of sodium bicarbonate was added withstirring continued for 1 hour. Acetone was removed under vacuum, and tothe residue was added 100 kg of water. The mixture was placed in arefrigerator overnight to give a white precipitate which was filtered toprovide 33 kg (wet) of the title compound.

Example 11

Synthesis of androst-5-en-17-one-3β,7β-diol: A 500 L reactor was charged230 kg methanol, 33 kg (wet) 3β-acetoxy-7β-hydroxy-5-androsten-17-one,108 kg water and 15 kg Na₂CO₃. The mixture was heated to reflux for 3hours. Methanol was removed under vacuum whereupon 250 kg of water wasadded to the residue. The mixture was put in refrigerator overnight togive a precipitate. The solids were collected by filtration, then washedwith water and dried at 40-50° C. to yield 9.5-10.5 kg (67.9-75.0%) ofthe title compound as a white solid.

Example 12

Purification of androst-5-en-17-one-3β,7β-diol: A 500 L reactor wascharged with 20 kg crude 3β, 7β-dihydroxyandrost-5-en-17-one and 200 kgmethanol and heated until all the solid dissolved. The solution wasfiltered while hot and after the filtrate cooled a white crystallinesolid formed. The solids were collected by filtration, washed with smallamount of methanol and dried at 40-50° C. The solid was then refluxed in50 kg of ethyl acetate for 20 minutes. After cooling the solid wasfiltered and dried at 40-50° C. under vacuum to provide 15.2 kg (76%) ofpurified title compound.

Example 13

Synthesis of 3β,7β-bis-(trimethylsiloxy)-5-androsten-17-one: A mixtureof 14.87 Kg of androst-5-en-17-one-3β,7β-diol, 23.8 Kg HMDS and 0.7 Kgsaccharin catalyst in 100 L acetonitrile was heated to reflux for 8hours with stirring under a nitrogen atmosphere. Liberated ammonia waspurged under slight vacuum. The reaction volume was then reduced bydistillation to collect 30 L of distillate (requires about 2 h). Thereaction volume was further reduced to half of the original reactionvolume by distillation under reduced pressure (700 mmHg), which requiresabout 2 h of heating at 50° C. The resulting uniform thick slurry iscooled to 5° C. (requires about 3 h), with additional acetonitrile addedto maintain a minimum mixing volume, and held at that temperature for 1.The precipitated product was collected by filtration and dried at 45-50°C. under vacuum (29 mmHg) to a loss on drying (LOD) of not more than 1%(requires 20 h) to provide 16 Kg (81% yield) of the title compound (95%purity).

Example 14

Synthesis of 17α-ethynyl-5-androstene-3β,7β,17β-triol: To 11.02 KgTMS-acetylene in 56.5 L tetrahydrofuran (THF) at −27° C. under anitrogen atmosphere was added 8.51 L 10M n-BuLi. The n-butyl lithium wasadded very slowly to maintain a temperature at −7 to −27° C. (requiresabout 2 h) and the resulting reaction was stirred 10 min. atapproximately 0° C. to produce TMS-lithium-acetylide. To theTMS-lithium-acetylide solution was added a solution of 25.41 Kg of3β,7β-bis-(trimethylsiloxy)-5-androsten-17-one in 95.3 L THF filteredthrough a 25 μm filter while allowing the reaction temperature to riseto 20-25° C. After addition was completed, the reaction temperature wasincreased to 40-45° C. To quench the reactor contents, 31.8 L ofmethanol was added over a period of about 1 h followed by 3.81 Kg KOH in18.4 L of water giving a final reactor temperature of 50° C. Liberatedacetylene is purged under slight vacuum. The reactor contents were thenconcentrated by distillation at 80° C. for 1 h then under vacuum (175mmHg) at about 70° C. (with an initial temperature of 25° C. to avoidbumping) to half of the original pot volume. The residue was cooled toabout 10° C. and 35.0 Kg of deionized water was added, followed by 16.4Kg 12N HCl while maintaining a pot temperature of about 10° C. andgiving a final pH of 1. Additional 26.0 kg deionized water was added andthe resulting mixture was stirred at about 5° C. for 1 h. The resultingslurry was filtered and washed with 75/25 mixture of methanol/water(16.9 L methanol, 5.6 L water). The collected solids were dried undervacuum (28 in Hg) at 45° C. for 12 h for a loss on drying of no morethan 0.5% to provide 9.6 Kg of the title compound (83% yield).

Example 15

Recrystallization of 17α-ethynyl-5-androstene-3β,7β,17β-triol: Crude 9.6Kg 17α-ethynyl-5-androstene-3β,7β,17β-triol prepared in Example 14 wasdissolved in refluxing 50/50 methanol/water (4.2 Kg methanol and 5.4 Kgwater). To the solution was added 33.4 Kg methanol followed by 37.6 Kgof THF. The mixture was heated to reflux and stirring was continueduntil all solids have dissolved, whereupon 99.8 Kg of deionized waterwas added while maintaining a reactor temperature of 60-75° C. Themixture was cooled to 0-5° C. over a period of 2 h and maintain at thattemperature for 1 h while stirring was continued. The solids wererecovered by filtration, washed with 9.6 Kg cold 50/50 methanol waterand dried under vacuum (28 in Hg) at 50° C. for 8 h to provide 8.2 Kg of17α-ethynyl-5-androstene-3β,7β,17β-triol. This first recrystallizationis used to remove trace colored impurities from the initial product. Asecond recrystallization was conducted by heating the solid from thefirst recrystallization in ˜10:1 methanol:water (145.8 Kg methanol and18.2 Kg of water) to 80° C. until all the solids have dissolved. Thesolution at 55-60° C. was filtered through a 25 μm filter to removeparticulate impurities, whereupon 2.5 Kg of methanol at 55-60° C. (usedto rinse the reactor) was added. Vacuum distillation at 125 mmHg at 70°C. was conducted until 0.9 to 1.2 times the volume of methanol that wasadded to the reactor was collected as distillate with water added asnecessary to permit stirring (about 120-160 Kg water added). Finalreaction volume was 200-225 L. The reactor mixture was cooled to 0-5° C.and maintained at that temperature for 1 h. The resulting slurry wasfiltered and the filter cake rinsed with 10 Kg deionized water and driedunder vacuum (28 in Hg) at 50° C. for 12 h to a residual water contentof less than 0.5%. This isolation procedure was used to reduce the THFcontent in the final product. The yield was 8.0 Kg of recrystallizedtitle compound (83% yield).

Example 16

Synthesis of 3β-acetoxy-androst-5-en-7-on-17-oxime:3β-Acetoxy-androst-5-en-7,17-dione (45 g, 130 mmol) was dissolved in 800mL methanol, 200 mL dichloromethane and 14.5 g Et₃N (144 mmol). To thesolution at RT was added a solution of 10 g of hydroxylaminehydrochloride dissolved in 200 mL methanol. After stirring overnight,200 mL of water was added followed by removal of volatile organics byevaporation under reduced pressure. To the resulting residue was addedan additional 1 L of water to give a while solid that was filtered andwashed well with water. Obtained was 45 g of crude title oxime in 95%purity by ¹H-NMR, which was used in the next step without furtherpurification.

Example 17

Synthesis of 3β-acetoxy-androst-5-en-17-oxime-7β-ol: To a solution of 44g of 3β-acetoxy-androst-5-en-7-on-17-oxime (100 mol %) in 800 mLmethanol and 200 mL tetrahydrofuran was added 50 g of cerium chlorideheptahydrate (110 mol %) in 20 mL of methanol. The resulting mixture wasstirred until the solids were completely dissolved. To the solutioncooled to about −5° C. was added 7 g sodium borohydride over 30 min.After stirring an additional 1.5 h at −5° C., the reaction mixture wasquenched with acetone (100 mL) and then allowed to warm to roomtemperature over a 30 min. period. The quenched reaction mixture wasconcentrated under vacuum to remove volatile organics. To the residuewas added 800 mL of water followed by extraction with ethyl acetate(3×500 mL). The combined organic extracts were washed with brine, driedover Na₂SO₄, then concentrated to provide 42 g of the title compound asa white foam, which was used in the next step without furtherpurification.

Example 18

3β-acetoxy-androst-5-en-17-one-7β-ol: To a solution of 42 g of3β-acetoxy-androst-5-en-17-oxime-7β-ol (100 mol %) in 200 mL of ethanolwas added 100 mL of water followed by 80 g (400 mol %) of sodiumdithionite. The reaction was heated at 55° C. and stirred 16 h. Aftercooling, the reaction was concentrated under reduced pressure. Theresidue was diluted with 100 mL of water, and the resulting solid wascollected by filtration and redissolved in 1 L dichloromethane. To theDCM solution was added 1 g activated carbon. After stirring overnightthe mixture was filtered, and the resulting filtrate was washed withwater, dried and concentrated to provide 25 g of crude product.Recrystallization from ethyl acetate gave 22 g of the title compound.

Example 19

Estrogen receptor binding assay: A suitable example system is anestrogen receptor-kit manufactured by PanVera for ERIβ, which containsrecombinant estrogen receptor β ligand, FLUORMONE™ ES2 (ES2), afluorescently labeled estrogen ligand, and appropriate buffer. Thesystem was used in a fluorescence polarization competition assay inwhich a test article, such as a preparation of Compound 1 or a positivecontrol displaces ES2 from its binding site. When bound to ERβ, ES2tumbles slowly and has a high fluorescence polarization value. UnboundES2 tumbles quickly and displays a low fluorescence polarization value.The change in polarization value in the presence of test compound thendetermines relative binding affinity of that test compound for ERβ asexpressed by its IC₅₀, which is the concentration of test compound thatresults in half-maximum shift in polarization. From IC₅₀, K_(i) wascalculated using the Cheng-Prusoff equation [Biochem. Pharmacol. 22:3099-3108, (1973)]: K_(i)=IC₅₀/(1+D/K_(d)) where D is the concentrationof ES2 and K_(d) is the dissociation constant for binding of ES2 to ERβ(K_(d)=4±2 nM).

The competition assay was conducted according to the manufacturer'sprotocol (Lit. No. L0712, Rev. 10/03). Assay reagents used werebacculovirus expressed, full length human ERβ 4.5 μmol/μL in 50 mMBis-Tris Propane (pH=9), 400 mM KCl, 2 mM DTT, 1 mM EDTA, 10% glycerol,ES2 400 nM in methanol and E2 screening buffer consisting of 100 mMpotassium phosphate (pH=7.4), 100 μg/mL BGG, 0.02% NaN₃. The ES2-ERβcomplex was formed with 20 μL 20 nM ERβ (0.020 pmol/μL) and 20 μL 2 nMES2 (0.002 pmol/μL). Positive control (estrogen) solution was preparedusing 20 μL of a 1.0 mM stock solution in DMSO and 80 μL DMSO. In afirst dilution, 50 μL of this solution is added to 50 μL of DMSO, whichis followed by dilutions in 2-fold increments, to provide for a 14 pointdilution curve. In a second dilution, to 4 μL of each DMSO solution fromthe first dilution is added 400 μL of ES2 screening buffer. To 20 μL oftest compound, serially diluted in the manner described immediatelyabove, in a 384 well black flat bottom microtiter plate, was added 20 μLof the ES2-ERI3 complex (0.5% final DMSO concentration) followed byincubation in the dark at 20-30° C. for 1-4 h. Test compound was treatedsimilarly except the starting concentration was 10 mM. Fluorescencepolarization values are obtained using 485 nm excitation and 530 nmemission interference filters. Binding assay for ERα was conducted asfor ERI3 except bacculovirus expressed, full length human 2.8 pmol/μLERα was used as reagent with the ERα-ES2 complex formed from 20 μL 30 nM(0.030 pmol/μL) and 20 μL 2 nM ES2 (0.002 pmol/μL).

Example 20

AR, GR and PR receptor binding assays. The AR competition assay wasconducted according to the manufacturer's protocol (Lit. No. L0844, Rev.05/02) in the manner described for ERβ with the following exceptions.Reagents used were recombinant rat androgen receptor ligand bindingdomain tagged with His and GST [AR-LBD (His-GST)] 0.38 pmol/μL in buffercontaining protein stabilizing agents and glycerol (pH=7.5), 200 nMFLUORMONE™ AL Green, which is a fluorescently labeled androgen ligand,in 20 mM Tris, 90% methanol and AR screening buffer containingstabilizing agents and glycerol (pH=7.5) with 2 of 1 mM DTT added per mLscreening buffer (AR screening buffer 2 mM in added DTT) was used as thereagents. The AL Green-AR complex was formed with 20 μL 50 nM AR (0.050pmol/μL) and 20 μL 2 nM AL Green (0.002 pmol/μL). K was calculatedusing, for the dissociation constant for binding of the fluorophore toreceptor, K_(d)=20±10 nM.

The PR competition assay was conducted according to the manufacturer'sprotocol (Lit. No. L0503, Rev. 06/03) in the manner described for ERβwith the following exceptions. Reagents used were recombinant humanprogesterone receptor ligand binding domain tagged with GST [PR-LBD(GST)] 3.6 pmol/μL in 50 mM Tris (pH=8.0), 500 mM KCl, 1M urea, 5 mMDTT, 1 mM EDTA and 50% glycerol, 400 nM FLUORMONE™ PL Green, which is afluorescently labeled progesterone ligand, in 20 mM Tris 90% methanol(pH=6.8) and PR screening buffer containing protein stabilizing agentsand glycerol (pH=7.4) with 4 μL of 1 mM DTT added per mL screeningbuffer (PR screening buffer 4 mM in added DTT). The PL Green-PR complexwas formed with 20 μL 80 nM PR (0.080 pmol/μL) and 20 μL 4 nM PL Green(0.004 pmol/μL). K_(i) was calculated using, for the dissociationconstant for binding of the fluorophore to receptor, K_(d)=40 nM.

The GR competition assay was conducted according to the manufacturer'sprotocol (Lit. No. L0304, Rev. 12/01) in the manner described for ERβwith the following exceptions. Reagents used were recombinant fulllength human glucocorticoid receptor 0.240 pmol/μL in 10 mM phosphatebuffer (pH=7.4), 200 mM Na₂MoO₄, 0.1 mM EDTA, 5 mM DTT and 10% glycerol,200 nM FLUORMONE™ GS1, which is a fluorescently labeled glucocorticoidligand, in 75% methanol, and GR screening buffer containing 100 mMpotassium phosphate (pH=7.4), 200 mM Na₂MoO₄, 1 mM EDTA, 20% DMSO with 5μL of 1 mM DTT per mL screening buffer added (GR screening buffer 5 mMin added DTT), 1 mM GR stabilizing peptide, which is a co-activatorrelated peptide [see Chang, C. Y. Mol. Cell Biol. 19: 8226-36 (1999)] in10 mM phosphate buffer (pH=7.4) and 1 M DTT in water were used as thereagents. To 2.5 mL of the GR screening buffer is added 2.5 mL GRstabilizing peptide solution and 125 μL of 1 M DTT to form the GRstabilizing peptide-glucocorticoid receptor complex. Order of additionto the microtiter plate was 20 μL test compound in 1% DMSO, 10 μL of 16nM GR (0.016 pmol/μL) and finally 10 μL of 4 nM GS1, followed byincubation in the dark at 20-30° C. for 4 h (total experiment timeshould not exceed 7 h). K_(i) was calculated using, for the dissociationconstant for binding of the fluorophore to receptor, K_(d)=0.3±0.1 nM.

Example 21

Impurity profiling of 17α-ethynyl-5-androstene-3β,7β,17β-triol(Compound 1) preparations.

Process A: HPLC conditions for Impurity profiling of Compound 1preparations form Process B are give in Table 1.

TABLE 1 HPLC Conditions for Impurity Profiling of Compound 1Preparations form Process A Column Waters XTERA ™ RP18, 3.5 μm, 4.6 mm(ID) × 150 mm (L) Mobile Phase A 100% Deionized water (degassed) MobilePhase B 100% Acetonitrile (degassed) Column Temperature 30° C. DetectionWavelength 210 nm Mobile Phase (initial) 90% Mobile Phase A; 10% MobilePhase B Flow Rate (initial) 1.0 mL/min Pump Gradient Program Time (min)% A % B Flow rate 0.00 90.0 10.0 1.00 40.0 20.0 80.0 1.00 43.00 20.080.0 1.00 43.01 90.0 10.0 1.00 50.00 (end) 90.0 10.0 1.00 InjectionVolume 10 μL Run Time 50 minutes

TABLE 2 Impurities in example preparation of Compound 1 preparedaccording to Process A, Route 1 before recrystallization Compound RRT*Peak Area % Unknown 0.63 0.59 Androst-5-en-17-one-3β,7β-diol 0.87 0.1217α-ethynyl-androst-5-ene- 1.00 96.58 3β,7β,17β-triol (Compound 1)17α-ethynyl-androst-5-ene- 1.04 0.99 3β,7α,17β-triol17α-ethenyl-androst-5-ene- 1.09 0.93 3β,7β,17β-triol17α-ethynyl-androst-5-en-7-one- 1.16 0.06 3β,17β-diol Unknown 1.47 0.04Unknown 1.60 0.06 17α-ethynyl-androst-5-ene- 1.75 0.63 3β,17β-diol*RRT-relative retention time (approximate) referenced to Compound 1(i.e., RRT of Compound 1 arbitrarily set to 1.00)

TABLE 3 Impurities identified in example preparation of Compound 1prepared according to Process A, Route 1 after recrystallizationCompound RRT* Peak Area % Unknown 0.55 0.13Androst-5-ene-3β,7β,17β-triol 0.87 0.06 17α-ethynyl-androst-5-ene- 1.0097.67 3β,7β,17β-triol (Compound 1) 17α-ethynyl-androst-5-ene- 1.05 0.723β,7α,17β-triol 17α-ethenyl-androst-5-ene- 1.09 0.65 3β,7β,17β-triol17α-ethynyl-androst-5-en-7-one- 1.16 0.05 3β,17β-diol Unknown 1.60 0.0517α-ethynyl-3β-acetoxy-androst-5-ene- 1.72 0.05** 17β-diol17α-ethynyl-androst-5-ene- 1.75 0.61** 3β,17β-diol *RRT-relativeretention time (approximate) referenced to Compound 1 (i.e., RRT ofCompound 1 arbitrarily set to 1.00) **Impurities with determined bindingcapacity to estrogen receptors (either directly or from the productderived after ester hydrolysis)

Process B: HPLC conditions for Impurity profiling of Compound 1preparations form Process B are identical to those of Table 1.

TABLE 4 Impurities identified in example preparation of Compound 1prepared according to Process B, Route 1 before recrystallizationCompound RRT Peak Area % androst-5-en-17-one-3β,7β-diol 0.94 0.8017α-ethynyl-androst-5-ene- 1.00 98.30 3β,7β,17β-triol (Compound 1)17α-ethynyl-androst-5-ene- 1.02 0.26 3β,7α,17β-triol17α-ethynyl-androst-5-en-7-one- 1.06 0.08 3β,17β-diol3β-acetoxy-androst-5-en-17-one-7β-ol 1.24 0.10 DHEA 1.27 0.033β-acetoxy-androst-5-en-17-one 1.46 0.093β-acetoxy-17-ethylenedioxy-androst-5-ene 1.50 0.123β,7β-bis-(trimethsilyloxy)-androst-5-en-17-one 1.68 0.21 *RRT-relativeretention time (approximate) referenced to Compound 1 (i.e., RRT ofCompound 1 arbitrarily set to 1.00)

Invention embodiments include the following. A process for preparationof a 17α-alkynyl-androst-5-ene-3β,7β,17β-triol essentially free ofsteroid side-product lacking an oxygen substituent at position 7comprising the steps of (a) contacting a suitably protecteddehydroepiandrosterone with an oxidizing agent to directly introduce the═O (ketone) functional group at position 7; (b) contacting a suitablyprotected androst-5-en-7,17-dione-3β-ol with a reducing agent to convertthe ═O (ketone) functional group at position 7 to hydroxyl predominatelyin the β-configuration; and (c) contacting an optionally protectedalkynyl anion with suitably protected androst-5-en-17-one-3β,7β-diol tointroduce an alkynyl substituent at position 17 predominately in theα-configuration by addition of the alkynyl anion to the ═O (ketone)functional group at position 17. The suitably protectedandrost-5-en-7,17-dione-3β-ol can have the structure

wherein R¹ is —H, C₁₋₆ alkyl, C₃₋₆ cycloalkyl, optionally substitutedaryl or optionally substituted heteroaryl and R² is —H, C₁₋₆ alkyl oraryl.

What is claimed is:
 1. A method to make17α-ethynyl-androst-5-ene-3β,7β,17β-triol essentially free of steroidside-product lacking an oxygen substituent at position 7 comprising thesteps of, (a) contacting a compound having the structure

with a reducing agent to convert the ═O functional group at position 7to hydroxy predominantly in the β-configuration wherein R¹ is —H, C₁₋₆alkyl, C₃₋₆ cycloalkyl, optionally substituted aryl or optionallysubstituted heteroaryl and R² is C₁₋₆ alkyl or aryl; (b) converting the═NOR² group at position 17 in the reaction product from step (a) to a ═Ogroup to obtain a 7β-hydroxy-17-one compound; (c) converting the—OC(O)R¹ group at position 3 of the 7β-hydroxy-17-one compound to a3β-hydroxy derivative of the 7β-hydroxy-17-one compound; (d) convertingthe hydroxy groups at positions 3 and 7 in the 3β-hydroxy derivative ofthe 7β-hydroxy-17-one compound from step (c) to (R³)₃SiO— groups,wherein each R³ independently is C₁₋₆ alkyl or aryl; and (e) contactingthe reaction product of step (d) with an compound having the structureof M-C≡C—Si(R⁵)₃, wherein R⁵ independently are C₁₋₆ alkyl or aryl and Mis a Group I, Group II or transition metal, wherein17α-ethynyl-androst-5-ene-3β,7β,17β-triol essentially free of17α-alkynyl-androst-5-ene-3β,17β-diol is obtained.
 2. The method ofclaim 1 wherein R¹ is C₁₋₆ alkyl or phenyl.
 3. The method of claim 2wherein R¹ is —CH₃.
 4. The method of claim 2 wherein R¹ and R² are —CH₃.5. The method of claim 1 wherein M is Li, Mg or Zn.
 6. The method ofclaim 1 wherein R⁵ is —CH₃.
 7. The method of claim 1 wherein M is Li andR⁵ is —CH₃.
 8. The method of claim 1 wherein comprising the additionalstep of purifying the 17α-ethynyl-androst-5-ene-3β,7β,17β-trial of step(e) by recrystallization.
 9. The method of claim 8 wherein R¹ is —CH₃.10. The method of claim 8 wherein R¹ and R² are —CH₃.
 11. The method ofclaim 8 wherein M is Li, Mg or Zn.
 12. The method of claim 8 wherein R⁵is —CH₃.
 13. The method of claim 8 wherein M is Li and R⁵ is —CH₃. 14.The method of claim 8 wherein the17α-ethynyl-androst-5-ene-3β,7β,17β-triol of step (e) is purified byrecrystallization from methanol-water.
 15. The method of claim 14wherein R¹ is —CH₃.
 16. The method of claim 14 wherein M is Li, Mg orZn.
 17. The method of claim 14 wherein R⁵ is —CH₃.